The sarcoplasmic recticulum (SR) is an internal membrane system in muscle, which functions as a Ca2+-sink during relaxation as as a Ca2+-source during contraction. Relaxation is mediated by the transport of Ca2+into the SR lumen by the Ca2+-ATPase (SERCA2), which is under regulation by phospholamban (PLB) in cardiac, slow-twitch skeletal and smooth muscles. Dephosphorylated PLB is an inhibitor of the affinity of the SR Ca2+-pump for Ca2+and phosphorylation relieves this inhibition. Alterations is in the expression levels of PLB or the SR Ca2+-ATPasehave been linked to altered Ca2+ homeostasis and deterioration of cellular function in several diseases. While transgenic mice have been rccently generated, which elucidated the functional role of altered PLB expression in vivo, focused on cardiac muscle and the physiological significance of PLB in other muscle and non-muscle tissues is not well understood. Thus, the objectives of the present proposal are to generate mouse models, with altered expressionof PLB or the SR Ca2+-ATPase to better define the function of each of these two key Ca2+-handling protein in vivo. Specifically, we will generate mice: a) overexpressing PLB and its phosphorylation mutants in either smooth or soleus muscle. Studies in these models coupled with studies in the PLB knockout mouse will elucidate the functional role of PLB in smooth and soleus muscles and define the second messanger pathways regulating these muscles through phosphorylation of PLB; b) overexpressing PLB in multiple tissues and under the control of an inducible promoter to achieve tight temporal and quantitative control of PLB expression in a reversible manner. These models will permit evaluation of the role of temporal alterations in PLB expression levels on cellular function; and c) overexpressing each of the SERCA2 isoforms (SERCA2a or SERCA2b) or conditionally ablating SERCA2 expression in a tissue specific manner. The models with altered SERCA2 expression levels will elucidate the role of this protein in the intact animal. Overall, the proposed animal models will provide valuable and unique systems for the biomedical community at large to carry out further studies on elucidating the functional role of PLB and SERCA2 in intracellular calcium handling in health and disease.